PYROSEQUENCING METHODS AND RELATED COMPOSITIONS
First Claim
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1. A method for determining the nucleotide sequence of a single-stranded DNA comprising performing the following steps for each nucleic acid residue of the DNA whose identity is to be determined:
- (a) contacting the DNA under DNA polymerization-permitting conditions with (i) a 3′
-O-blocked dNTP selected from the group consisting of 3′
-O-blocked dATP, 3′
-O-blocked dCTP, 3′
-O-blocked dGTP, and 3′
-O-blocked dTTP, and (ii) 9°
N DNA polymerase (exo-) A4851/Y409V;
(b) (i) determining whether pyrophosphate is generated as a result of step (a), whereby (1) pyrophosphate generation indicates that polymerization has occurred and the identity of the nucleic acid residue in the DNA is that which is complementary to the 3′
-O-blocked dNTP used in part (i) of step (a), and (2) the absence of pyrophosphate generation indicates that the identity of such nucleic acid residue is not that which is complementary to such 3′
-O-blocked dNTP, and (ii) if pyrophosphate is not generated, repeating step (a) once, twice or three times as necessary, wherein in each repetition a 3′
-O-blocked dNTP is used which is different from any 3′
-O-blocked dNTP already used, and determining, after each repetition of step (a), whether pyrophosphate is generated, such generation indicating that polymerization has occurred and the identity of the nucleic acid residue in the DNA is that which is complementary to the 3′
-O-blocked dNTP used in part (i) of the repeated step (a); and
(c) removing from the 3′
-O-blocked dNTP polymerized in step (a) or (b), whichever is applicable, the moiety blocking the 3′
-O atom of the dNTP, with the proviso that such removing step is optional in the event that there remains no further nucleic acid residue of the DNA whose identity is to be determined.
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Abstract
This invention provides methods for pyrosequencing and compositions comprising 3′-O-modified deoxynucleoside triphosphates.
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Citations
55 Claims
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1. A method for determining the nucleotide sequence of a single-stranded DNA comprising performing the following steps for each nucleic acid residue of the DNA whose identity is to be determined:
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(a) contacting the DNA under DNA polymerization-permitting conditions with (i) a 3′
-O-blocked dNTP selected from the group consisting of 3′
-O-blocked dATP, 3′
-O-blocked dCTP, 3′
-O-blocked dGTP, and 3′
-O-blocked dTTP, and (ii) 9°
N DNA polymerase (exo-) A4851/Y409V;(b) (i) determining whether pyrophosphate is generated as a result of step (a), whereby (1) pyrophosphate generation indicates that polymerization has occurred and the identity of the nucleic acid residue in the DNA is that which is complementary to the 3′
-O-blocked dNTP used in part (i) of step (a), and (2) the absence of pyrophosphate generation indicates that the identity of such nucleic acid residue is not that which is complementary to such 3′
-O-blocked dNTP, and (ii) if pyrophosphate is not generated, repeating step (a) once, twice or three times as necessary, wherein in each repetition a 3′
-O-blocked dNTP is used which is different from any 3′
-O-blocked dNTP already used, and determining, after each repetition of step (a), whether pyrophosphate is generated, such generation indicating that polymerization has occurred and the identity of the nucleic acid residue in the DNA is that which is complementary to the 3′
-O-blocked dNTP used in part (i) of the repeated step (a); and(c) removing from the 3′
-O-blocked dNTP polymerized in step (a) or (b), whichever is applicable, the moiety blocking the 3′
-O atom of the dNTP, with the proviso that such removing step is optional in the event that there remains no further nucleic acid residue of the DNA whose identity is to be determined. - View Dependent Claims (38, 39, 41, 42, 43, 45, 53, 54, 55)
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2-37. -37. (canceled)
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46. A compound comprising a dNTP having bound to its 3′
- oxygen an allyl moiety, a methoxymethyl moiety, an allyl derivative or a methoxymethyl derivative.
- View Dependent Claims (47, 48, 49)
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50. A process for producing a 3′
- -O-allyl dNTP comprising;
(a) sequentially contacting a dimethoxytrityl (DMTr) 3′
protected nucleoside triphosphate with (i) 3-bromo propene, NaOH and benzene, and (ii) a suitable solvent; and(b) sequentially contacting the product of step (a) with (i) POCl3/(MeO)3P(O), (ii) tributylammonium pyrophosphate, and (iii) TEAB/NH4OH, so as to produce the 3′
-O-allyl dNTP. - View Dependent Claims (51, 52)
- -O-allyl dNTP comprising;
Specification